Nutraceutical Effect of Vitamins and Minerals on Performance And

Journal of Thermal Biology 84 (2019) 451–459

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Journal of Thermal Biology

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Nutraceutical eﬀect of vitamins and minerals on performance and immune and antioxidant systems in dairy calves during the nutritional transition T period in summer

Rael Bordignona, Andreia Volpatoa, Patrícia Glombowskya, Carine F. Souzab, Matheus D. Baldisserac, Rodrigo Seccod, Wanderson A.B. Pereirad, Marta L.R. Leale, Marcelo Vedovattof, Aleksandro S. Da Silvaa,b,* a Graduate Program of Animal Science, Universidade do Estado de Santa Catarina, Chapecó, Brazil b Graduate Program of Toxiciological Biochemistry, Universidade Federal de Santa Maria, Santa Maria, Brazil c Graduate Program of Pharmacology, Universidade Federal de Santa Maria, Santa Maria, Brazil d Veterinary Medicine, Instituto Federal Catarinense, Concordia, Brazil e Department of Large Animals, Universidade Federal de Santa Maria, Santa Maria, Brazil f Range Cattle Research and Education Center, University of Florida, Ona, FL, USA

ARTICLE INFO ABSTRACT

Keywords: We aimed to determine whether the use of injectable vitamins and minerals improves growth performance and Antioxidant system immune and antioxidant responses in dairy calves during pre- and post-weaning period in summer. Twenty dairy Calves calves (45 days of age) were randomized to two groups (10 each): control group (CON) and treated group ff Metaphylactic e ect [TREAT; injection providing 0.20, 0.80, 0.20, 0.10, 35 and 1 mg/kg of copper, zinc, manganese selenium, and Performance vitamins A and E, during two periods (15 days pre- and 15 days post-weaning)]. The animals were weighed and Immunity blood samples were collected on days 1, 15, 30 and 45 of the study. Levels of serum copper, selenium, zinc, and Oxidative stress fi Thermal stress manganese were measured on day 1; and the results showed that calves were not de cient in these minerals. The TREAT group had greater BW gain during the final third of the experiment. There was an increase in total leukocyte numbers as a result of elevation in neutrophil counts (day 45) and monocytes (days 30 and 45) in the TREAT group. This group also had lower reactive oxygen species (ROS) content (days 15, 30 and 45) and lipid peroxidation (LPO; days 15 and 45). Furthermore, the TREAT group had greater antioxidant capacity against peroxyl radicals (ACAP; days 15 and 30), activities of the enzymes glutathione peroxidase (GPx; days 15, 30 and 45) and superoxide dismutase (SOD; day 15), concentrations of total serum proteins (day 30), serum globulin (days 15 and 30), ceruloplasmin (day 15), tumor necrosis factor-alpha (TNF-α), interleukin-1, (IL-1; days 30 and 45) and interferon gamma (IFNγ; day 45), compared to CON group. High respiratory rates during hot times of the day in all study calves was suggestive of heat stress. Taken together, the data suggest that mineral and vitamins injections increased the growth performance and boosted the antioxidant and immunological systems of dairy calves during the diet transition period in summer.

1. Introduction Another challenge for calves is weaning. During this critical period, there are drastic changes related primarily to the transition from a li- The initial phase of cattle life is critical because their immune quid to a solid diet. Reduction in the levels of supplied dry matter and system is immature shortly after birth; nevertheless, the animal requires greater ruminal activity can aﬀect animal development (Campos and rapid development of the immune system in order to respond the in- Lizieire, 2000). According to the literature, this phase generates stress, fections and to optimize growth (Besser and Gay, 1994; Botteon et al., a factor often associated with reduced resistance to diseases (Grandin 2008). Therefore, these animals are dependent on the immunity ac- and Gallo, 2007). In addition, possible changes of facilities and man- quired from colostrum to generate immune responses, despite the fact agement (e.g., dehorning) commonly practiced during this period, can that they are capable of mounting some immune responses in utero. render the animal more vulnerable (Campos and Lizieire, 2000).

* Corresponding author. Aleksandro Schafer Da Silva Department of Animal Science, Universidade do Estado de Santa Catarina (UDESC), Chapecó, SC, Brazil. E-mail address: [email protected] (A.S. Da Silva). https://doi.org/10.1016/j.jtherbio.2019.07.034 Received 15 April 2019; Received in revised form 27 July 2019; Accepted 27 July 2019 Available online 29 July 2019 0306-4565/ © 2019 Elsevier Ltd. All rights reserved. R. Bordignon, et al. Journal of Thermal Biology 84 (2019) 451–459

Table 1 Calf diet during the experimental period, as chemical composition of hay and commercial concentrate used in animal feeding.

Ingredients Day 1–15 of experiment (preweaning) Day 15–30 of experiment (post-weaning) Day 30–45 of experiment (post-weaning)

Milk cow (liter/animal/day) 4.0 0 0 Commercial concentrate* (grams/animal/day 400 600 800 Hay# (grams/animal/day) Ad libitum Ad libitum Ad libitum

Chemical composition,a, # Hay# Concentratea

Dry matter (%) in dry matter 95.5 89.0 Mineral (%) in dry matter 7.5 7.3 Crude protein (%) in dry matter 12 16 Ethereal extract (%) in dry matter 1.4 2.8 Neutral detergent ﬁber (%) in dry matter 61 49.4 Acid detergent ﬁber (%) in dry matter 36.2 10.8 Mineral composition,a, # Copper (mg/kg) nd 37.6 Zinc (mg/kg) nd 136.2 Selenium (mg/kg) nd 0.85 Manganese (mg/kg) nd 41.8 Note: not-detected (nd). a Commercial concentrated guaranteed levels: calcium (Min/Max) 10–15 g/kg; phosphorus (Min) 5000 mg/kg; zinc (Min) 140 mg/kg; cobalt (Min) 2 mg/kg; copper (Min) 40 mg/kg; sulfur (Min) 2000 mg/kg; iodine (Min) 2 mg/kg; magnesium (Min) 1500 mg/kg; manganese (Min) 40 mg/kg; selenium (Min) 1 mg/kg; sodium (Min) 2000 mg/kg; Fluorine (Min) 100 mg/kg; virginiamycin (Min) 50 mg/kg; Vitamin A (Min) 10,000 IU/kg; Vitamin D3 (Min) 2000 IU/kg; and VitaminE (Min) 50 IU/kg.

The metabolic stresses associated with weaning can be severe, of dairy calves during the diet transition period (weaning) in summer. compromising the antioxidant system (Sundrum, 2015) and generating oxidative stress. This biochemical event is characterized by an im- 2. Materials and methods balance between the production of reactive oxygen species (ROS) and the exhaustion or activation of the animal's antioxidant system (Persson 2.1. Products et al., 2014; Vedovatto, 2018) possibly favoring deleterious effects on cells, tissues and consequently the onset of diseases (Burke et al., 2009). The commercial product used contained vitamins A and E Intake of a diet rich in antioxidants had beneficial effects on animal (Adaptador® Vit; Biogénesis Bagó, Buenos Aires, Argentina), and the health (López-Alarcón and Denicola, 2013). minerals zinc, copper, selenium, and manganese (Adaptador® Min; According to researchers, supplementation with minerals plays an Biogénesis Bagó). The dose used was 1 mL/50 kg of body weight, ac- important role in cattle by stimulating the immune system (Rink, cording to the manufacturer's recommendations. The dose of 2000), because many minerals are enzymatic cofactors (Filappi et al., Adaptador® Min provided 0.20 mg/kg of copper, 0.80 mg/kg of zinc, 2005). Recently, studies showed that injectable minerals would be a 0.20 mg/kg of manganese and 0.10 mg/kg of selenium. The dose of suitable method to improve mineral utilization by animals, and this Adaptador® Vit provided 35 mg/kg of vitamin A and 1 mg/kg of vi- may be a promising alternative to improve animal performance (Collet tamin E. On experimental days 1 (15 days pre-weaning = 45 days of et al., 2017). In addition, vitamins A and E were identified as anti- age) and 30 (15 days post-weaning = 75 days of age), was applied the oxidants (Sies, 1991). These are important for promoting good health, supplement subcutaneously, with the two products being applied alone and are key factors for calf-rearing and future performance (Weiss, and in different places. In the control group, the same management and 2002). application procedure was performed; however, the same volumes of Calves exhibit poor growth performance, low immunity, increased the treated group were applied with only saline solution (0.9% NaCl) respiratory rates, and greater susceptibility to diseases attributable to and mineral oil for purposes of the placebo effect. reduced feed intake during the summer months (West, 2003; Tao and Dahl, 2013; Kargar et al., 2018). The consequences of thermal stress are economic losses associated with reduced weight gain, and increased 2.2. Animals and experimental design mortality and morbidity (Roland et al., 2016). In a recent study, researchers observed that chromium supplementation in liquid and solid The study was carried out in a commercial farm in the western re- feeds improved growth performance in summer-exposed calves as a gion of Santa Catarina state, Brazil, using 20 Holstein heifer calves result of reduced respiration rate and increased feed intake (Kargar (45 ± 2 days of age) divided into two groups (10 animals each): et al., 2018). Studies from our research group showed that supple- control group (CON) and treated group (TREAT). The groups were mentation with minerals and/or vitamins in calves (Glombowsky et al., randomized in 20 pens, in order to randomize the effect of the en- 2018; Tomasi et al., 2018; Volpato et al., 2018) and lambs (Cazarotto vironment inside the shed during lactating phase. The animals were et al., 2018) are beneficial to the health and growth performance of the housed in individual pens at the beginning of the experiment because animals during the nursing phase. We also observed similar results they were nursing (between 45 and 60 days of age); however, at when minerals (zinc and selenium) were given during the transition weaning (60 days of age), they were allocated to four collective pens period from liquid to solid diet (weaning) in calves (Volpato et al., with five calves each, separated by groups. The four pens were located 2018). Our hypothesis was that the combination of injectable minerals next to each other, in the middle of the shed, and the groups were and vitamins would increase growth performance, and would augment distributed in the following order: CON-TREAT–CON–TREAT. Before immunological and antioxidant responses in dairy calves even in hot the study, the animals received colostrum (4 L/animal until 6 h after seasons. Therefore, the objective of this study was to determine whether birth), transition milk (4 L/animal/day until 4 days after birth) and the subcutaneous application of minerals and vitamins has nu- milk (4 L/animal/day until 60 days after birth). Up to 45 days after traceutical effects on antioxidants and consequent growth performance birth, the calves received water, hay (Cynodon spp.) and concentrate ad libitum (Table 1). Forty-five days after birth, the study started (day 1),

452 R. Bordignon, et al. Journal of Thermal Biology 84 (2019) 451–459 and calves received milk [only from day 1 to day 15 (weaning); 4 L/ of 5, 10, 15, 20, 40, 60, and 80 μmol/L Cu, Mn, and Zn. Sample digests animal/day], concentrate (day 1–15: 400 g/animal/day; day 15–30 of were diluted in order to present suitable concentrations for measure- 600 g/animal/day; day 30–45: 800 g/animal/day), water and hay ments. Recovery tests were also performed for sample digests. After (Cynodon spp.) ad libitum (Table 1). The liquid and solid feeds were each ten measurements, two standard Cu, Mn, and Zn solutions were provided in two meals (08:00 h and 16:00 h). Dehorning and im- analyzed to check the slope of calibration curve. In case of a slope munization were not done until the end of the study to avoid affecting difference of higher than 10%, the calibration curve was prepared again the serum variables. using all standards. All samples were assayed in triplicate. The experiment was carried out in the south of Brazil, in a shed without air conditioning, with lateral openings. The experiment took 2.5. Hematological analysis place during the summer months (January and February) and the temperature was measured inside the building during the day using an Blood stored in EDTA tubes was used to perform complete blood inside digital thermometer. Data of ambient temperature in the muni- counts. The erythrocyte count, total leukocytes and hemoglobin con- cipality of the experiment were obtained from a meteorological station centration were measured using semi-automatic equipment (CELM located 3.8 km away from the experimental shed; the minimum and model CC530). For the determination of hematocrit values, we used maximum temperatures recorded were 29 °C and 37 °C, respectively. capillary tubes, centrifuged for 1 min at 1400 rpm. At sampling, blood The hottest temperature occurred around 14:30 h throughout the ex- smears were performed and stained with commercial dye (Panótipo perimental period, and temperatures registered inside the shed at this Rápido) to perform leukocyte differential counts using a light micro- time of day temperatures were as high as 39.8 °C. scope at 1000× magnification (Feldman, 2000), with 100 cells ran- During the hottest times of the day, all animals increased their domly identified per slide. Knowing the number of total leukocytes breathing rates and panting in order to dissipate the heat. At the end of (μL), differential leukocytes (%) were calculated using absolute num- the experiment (day 42 of the experiment), between 08:00 h and bers of neutrophils, lymphocytes, monocytes and eosinophils. Counts 09:00 h and between 14:00 h and 15:00 h, the respiratory movements were expressed per microliter (μL) of blood. per minute were recorded for all heifers. 2.6. Analysis of oxidant/antioxidant status 2.3. Sample collection Serum ROS levels were determined using the 2′,7′-dichloro- The animals were weighted and feces and blood samples were col- fluorescein (DCF) oxidation method described by LeBel et al. (1992). lected on days 1 (15 days pre-weaning), 15 (weaning day: calves at 60 Fluorescence was measured using excitation and emission wavelengths days of age), 30 (15 days post-weaning) and 45 (30 days after weaning). of 485 and 538 nm, respectively. A calibration curve was established Blood samples were collected from jugular veins into two vacuum with standards of DCF (0.1 nM–1 μM), and results were expressed as U tubes, with and without anticoagulant (EDTA 10%). The samples were DCF/mg of protein. maintained in an isothermal box at 10 °C until the time of the labora- The methodology of lipid peroxidation (LPO) was based on Hermes- tory analysis. Samples collected in tubes without anticoagulant (clotted Lima et al. (1995) with some modifications by the authors, called FOX blood) were centrifuged at 7000 rpm for 10 min to separate serum. The based on the oxidation of Fe(II) under acidic conditions. The Fox animals were weighed using a tape measure, a method of weighing the method measures lipid peroxides, one of the principal products of lipid animals indirectly using thoracic perimeter (Reis et al., 2008). We also peroxidation. For LPO measurements, FeSO4 (1 mM), H2SO4 (0.25 M), performed fecal score analyses (Larson et al., 1977) on feces collected xylenol orange (1 mM, Sigma) and MilliQ water were sequentially from the rectal bulb. Fecal samples were kept in isothermal boxes at added. Samples or methanol (blanks) were added and incubated for 10 °C for further parasitological analysis. Concentrate and hay samples 30 min. Thereafter, absorbance (550 nm) was determined and cumene were collected from the total diet provided (concentrate and hay) for all hydroperoxide (CHP; Sigma) was employed as a standard. LPO was calves in the three periods (Table 1). We measured dry matter, ash, expressed as μmol CHP/mg protein. ether extract and crude protein, following a method described by Silva Total antioxidant capacity against peroxyl radicals (ACAP) was and Queiroz (2006). In addition, neutral detergent fiber and acid de- determined according to the method described by Amado et al. (2009). tergent fiber were analyzed following the methodology described by This method consists of determining the antioxidant capacity of tissues Van Soest (1994). Minerals (copper, zinc, selenium and manganese) using a fluorescent substrate (2′,7′ dichlorofluorescein diacetate - were determined in hay and concentrate by a near-infrared spectro- H2DCF-DA) and the production of peroxyl radicals by thermal de- scopy method in a commercial laboratory (Shankar, 2015, Table 1). composition of ABAP (2.2′-azobis 2 methylpropionamidine dihy- drochloride). The fluorescence was determined using a microplate 2.4. Serum concentrations of minerals reader (Spectramax I3), at 37 °C (excitation: 485 nm; emission: 530 nm) with readings at every 5 min over 30 min. The results were expressed as Serum concentrations of selenium, copper, manganese and zinc relative area (the difference between the area with and without ABAP were determined on day 1 of the experiment. In the analyses, specific divided by the area without ABAP), and levels were expressed as UF/ tests were used, as described below. Selenium levels were measured mg protein. using the hydride generation atomic absorption spectrometry technique The activity of superoxide dismutase (SOD) was determined ac- (HG-AAS) (PerkinElmer Model 3030), using chemicals of analytical cording to the auto-oxidation principle of pyrogallol, inhibited in the grade from Merck (Darmstadt, Germany; Cazarotto et al., 2018; Flores presence of SOD. The optical density change was determined kinetically et al., 2001). Thus, 1 mL of HNO3 and 250 μLH2O2 were added to for 2 min at 420 nm, at 10-s intervals according to the methodology 500 μL of serum into 15 mL vials. The vials were warmed in a com- described by Beutler (1984). The activity was expressed as U SOD/mg mercially-available microwave for 5 min. Milli-Q water was added until of protein. 10 mL final volume and the solution was analyzed. Copper, manganese, The activity of glutathione peroxidase (GPx) was measured in- and zinc concentrations in digested samples were determined using ICP- directly by monitoring the oxidation rate of NADPH at 340 nm using OES. Operational conditions used for these minerals determinations cumene hydroperoxide (CuOOH), according to Wendel (1981). The were those recommended by the instrument manufacturer. Aqueous enzymatic activity was expressed as U GPx/mg protein. calibration standards were prepared by sequential dilution of a stock The protein concentrations in serum were determined using the solution of copper, manganese, and zinc (10 μmol/L, Spex CertiPrep, Coomassie blue method following the methodology described by Read Metuchen, NJ, USA). Standards were prepared daily at concentrations and Northcote (1981), using bovine serum albumin as a standard.

453 R. Bordignon, et al. Journal of Thermal Biology 84 (2019) 451–459

Table 2 Serum levels of minerals copper, zinc, selenium and manganese in calves of the control and treated groups on days 1 of the experiment (45-day-old animals = 15 days pre-weaning).

Mineral Control (n =10) Treated (n =10) aP-value bvalues (min. – max.)

Copper (μmol/L) 14.8 ± 3.9 15.1 ± 2.8 0.841 2.91 to 19.65 Zinc (μmol/L) 25.1 ± 5.4 23.8 ± 4.7 0.802 5.46 to 36.70 Manganese (μmol/L) 0.85 ± 0.05 0.87 ± 0.07 0.910 0.40 to 1.00 Selenium (μg/L) 80.4 ± 7.0 76.0 ± 10.3 0.751 51.0 to 85.0

a No statistical difference between groups (P > 0.05). b Studies have reported minimum and maximum levels for cattle for minerals: copper and zinc (Pavlata et al., 2005), selenium (Villard et al., 2002), and manganese (Jokubauskienė et al., 2010). Note: Pavlata et al. (2005) defined as copper and zinc deficiency in blood serum concentrations of the respective element below 12 μmol/L.

2.7. Proteinogram when needed. Two-way ANOVA was performed using repeated measurements to test for differences in the parameters over time (con- For protein fractionation, polyacrylamide gel electrophoresis with sidering blocks of groups CON and TREAT) and compared between sodium dodecyl sulphate (SDS-PAGE) was performed according to a groups (controlling data dependency due to dependence in time). technique described by Fagliari et al. (1998) using mini-gels Significant difference was set at P < 0.05. Statistical manipulations (10 × 10 cm). The gels were stained with Coomassie blue and photo- were performed using R-language, v 3.1 (R Development Core Team, graphed to identify and quantify protein fractions using Labimage1D 2012). software (Loccus Biotechnology). Standards containing fractions with molecular weights between 10 and 250 kDa (Kaleidoscope - BIORAD) 3. Results were used as reference for the identification of protein fractions. 3.1. Serum mineral concentrations 2.8. Cytokines On day 1 of the experiment, there were no significant differences Cytokine quantification (tumor necrosis factor-alpha - TNF-α, in- between groups; furthermore, values of copper, zinc, manganese and terleukin-1, IL-1 and interferon gamma – IFNγ) was assessed by ELISA selenium were within the reference values for calves with no defi- using commercial Quantikine immunoassay kits according to the ciencies in these minerals (Villard et al., 2002; Pavlata et al., 2005; manufacturer's instructions. Briefly, 96-well microplates were sensi- Jokubauskienė et al., 2010, Table 2). tized with primary antibody at room temperature for 30 min; samples were added and incubated for 30 min at 37 °C. After washing, secondary 3.2. Growth performance and clinical signs antibodies conjugated with peroxidase were added to each well and incubated. The concentration of the cytokines was determined by the There were no significant differences in fecal parameters (color, intensity of the color measured spectrophotometrically using a micro- fluidity, odor or consistency) between groups (P = 0.854; data not plate reader. shown). However, it should be noted that the vast majority (more than 95%) of the animals had feces with scores within the normal range. 2.9. Fecal score and parasitological examination Feces of runny and (4) watery consistency were not observed, and feces with soft consistency (but with healthy aspect) were observed rarely in The occurrence of diarrhea was observed daily following the both groups at approximately 48 h after the withdrawal of milk as feed, methodology described by Larson et al. (1977), which is based on fecal lasting 2–3 days. On the day of blood collection, all calves had normal score and fluidity: (1) normal; (2) soft; (3) runny and (4) watery. For feces. All animals were negative for parasites during the experimental the determination of parasitological infection, the technique described period. by Monteiro (2010) was used with subsequent reading using light mi- The TREAT group had greater BW gain during the final third of the croscopy. Fecal consistency and parasitic infection were evaluated only experiment (days 15–45; and days 30–45 of the experiment) compared to monitor the health of calves because our goal was to use only healthy to the CON group (Table 3). Over time, the BW of animals from both calves to evaluate the effect of minerals and vitamins. groups increased (Table 3). The respiratory rates increased during the hottest times of the day, 2.10. Statistical analysis i.e. in the morning (0800 h–0900 h). We observed that 26 and 40 breaths/minute (mean CONT was 28.5, and TREAT was 29.2) and in Data were analyzed using descriptive statistics for contingency of the afternoon (1400 h–1500 h) varied between 39 and 52 breaths/ information and for further assumptions that were presented as de- minutes (mean CONT was 46.6, and TREAT was 43.8). There was no scriptive (mean and standard deviation) for blood cell parameters: he- difference between groups (P > 0.05) at both moments. matocrit, erythrocyte count, hemoglobin, leukocytes, lymphocytes, monocytes and eosinophils. The second set of data were for GPx and 3.3. Hemogram SOD activities, followed by biochemical components: ROS, LPO, and ACAP. The third group of parameters measured were proteinogram The results of hematological analyses are shown in Table 4.No (total protein, globulin, albumin, ceruloplasmin, and immunoglobulins) differences were observed between groups and over time in terms of and cytokines (TNF-α, IL-1, and IFNγ). Finally, we took measurements erythrocyte numbers, hematocrit and hemoglobin concentrations. A associated with animal weight: body weight and weight gain. The Chi- greater number of total leukocytes (P = 0.050) was observed in the square test was used to evaluate fecal score. Each calf was considered animals of the TREAT group on day 45, as a consequence of the in- an experimental unit. For each group (CON and TREAT) and day of creased neutrophil counts (P = 0.023). The number of monocytes was observation (days 1, 15, 30, and 45), all parameters were tested for greater in the TREAT group on days 30 and 45 (P = 0.045 and 0.037, normality using the Shapiro-Wilk test. Skewness, kurtosis and homo- respectively). The number of lymphocytes and eosinophils did not differ geneity were evaluated using the Levene test, or log transformation between groups (Table 4).

454 R. Bordignon, et al. Journal of Thermal Biology 84 (2019) 451–459

Table 3 Over time, the number of leukocytes and neutrophils increased only Body weight and weight gain of dairy calves that received mineral and vitamin in the TREAT group (P = 0.044 and 0.001, respectively). The other applications (treated group) by the subcutaneous route on days 1 (15 days pre- hemogram variables did not diﬀer over time in either group. weaning) and 30 days (15 days post-weaning) of the experiment.

Variable Day Control (n = 10) Treated (n = 10) P-value 3.4. Oxidant and antioxidant status

c c Body weight (kg) 1 58.6 (8.9) 61.3 (9.4) 0.847 Oxidative and antioxidant status results are shown in Fig. 1. The 15 72.3 (15.5)bc 72 (13.2)bc 0.923 30 90.4 (17.4)ab 91.2 (15)b 0.904 TREAT group showed lower levels of ROS on days 15, 30 and 45 45 108.7 (14)a 120.7 (16.7)a 0.187 (P = 0.041, 0.001 and 0.001, respectively), as well as lower levels of P-value 0.001 0.001 LPO on days 15 and 45 (P = 0.050 and 0.040, respectively). ACAP le- Weight gain (kg) 1–15 13.7 (4.6) 10.7 (4.2) 0.845 vels in the TREAT group had greater values on days 15 and 30 – 1 30 31.8 (8.7) 29.9 (8.1) 0.695 (P = 0.001 and 0.001, respectively). The GPx activity was greater on 1–45 50.1 (8.2) 59.4 (7.3) 0.085 15–30 18.1 (5.3) 19.2 (4.1) 0.745 days 15, 30 and 45 in the TREAT group (P = 0.001, 0.001 and 0.050, 15–45 36.4 (8.9) 48.7 (7.0) 0.050* respectively), while SOD activity was greater only on day 15 of the 30–45 18.5 (4.6) 29.5 (7.6) 0.024* experiment (P = 0.036). Over time, there was a reduction in ROS levels in the TREAT group, Note: results presented in mean and standard deviation. P ≤ 0.05 (*) on the i.e. days 1, 15 and 30 to day 45 (Fig. 1). LPO levels were lower in both same line shows the differences between groups. P ≤ 0.05 in the same column groups, i.e. day 1–45 (Fig. 1). The application of minerals and vitamins shows the differences over time in each group, the differences being represented by different letters. generated an increase of ACAP, SOD and GPx over time (Fig. 1) that did not occur in the CON group. Table 4 Hemogram of dairy calves that received mineral and vitamin applications 3.5. Proteinogram (treated group) by the subcutaneous route on days 1 (15 days pre-weaning) and 30 days (15 days post-weaning) of the experiment. Total protein levels were greater (P = 0.001) in the TREAT group on day 30, as a consequence of the increase in globulin levels on days Variable Day Control (n = 10) Treated (n = 10) P-value 15 and 30 (P = 0.020 and 0.035, respectively; Fig. 2). Levels of albumin Erythrocytes (x106 μL) 1 4.2 (1.08) 4.6 (0.78) 0.598 did not differ between groups and over time (Fig. 2). The proteinogram 15 4.25 (0.92) 4.12 (0.56) 0.746 revealed that globulins were elevated due to increased levels of IgA 30 5.24 (0.75) 4.52 (0.64) 0.114 (days 15 and 30; P = 0.050 and 0.044, respectively), IgG heavy chain 45 4.59 (0.85) 5.34 (0.80) 0.775 (days 15, 30 and 45; (P = 0.031, 0.001 and 0.013, respectively) and P-value 0.285 0.062 Hemoglobin (g/dL) 1 7.94 (1.27) 9.08 (0.57) 0.201 ceruloplasmin (day 15; P = 0.001) (Fig. 2). 15 8.68 (0.72) 9.27 (1.02) 0.463 Over time, only animals in the TREAT group showed increased total 30 9.34 (0.83) 8.60 (0.97) 0.495 protein (day 1–30), globulin (day 1–30), IgG heavy chains (day 1–45), 45 9.63 (1.17) 9.47 (0.57) 0.802 IgA (days 15 and 30 to day 45) and ceruloplasmin (day 15–45) levels P-value 0.294 0.376 ff Hematocrit (%) 1 32.1 (5.1) 36.4 (3.8) 0.408 (Fig. 2). In CON animals, there were no di erences over time. 15 32.7 (4.0) 32.9 (5.6) 0.864 30 37.9 (4.5) 34.3 (4.5) 0.653 3.6. Cytokines 45 36.4 (4.0) 35.8 (3.5) 0.906 P-value 0.571 0.789 Results of cytokine levels are shown in Fig. 3. In the calves in the Leukocytes (x103 μL) 1 8.35 (1.9) 9.68 (2.2)ab 0.569 b α 15 9.63 (2.0) 8.41 (1.9) 0.528 TREAT group, we observed that there were increases in serum TNF- 30 10.0 (3.1) 9.12 (3.8)ab 0.635 and IL-1 levels on days 30 (P = 0.001 and 0.001, respectively) and 45 45 9.18 (1.2) 11.6 (1.8)a 0.050* (P = 0.001 and 0.001, respectively) of the experiment, as well as IFNγ P-value 0.208 0.044* 3 μ a on day 45 (P = 0.027). Over time, only animals in the TREAT group Neutrophils (x10 L) 1 3.95 (1.22) 4.35 (1.4) 0.756 α 15 4.81 (1.42) 3.89 (1.50) b 0.305 showed increases in TNF- (day 1 to days 30 and 45), IL-1 (day 1 to 30 4.42 (1.83) 5.29 (2.51) ab 0.652 days 30 and 45) and IFNγ (days 1 and 15 to day 45) levels (Fig. 3). 45 3.97 (1.30) 6.13 (2.17)a 0.023* P-value 0.598 0.001* 4. Discussion Lymphocytes (x103 μL) 1 4.03 (0.98) 5.02 (1.73) 0.422 15 4.48 (1.46) 4.31 (1.43) 0.654 30 5.41 (1.60) 3.43 (1.30) 0.235 The application of injectable minerals and vitamins ensures a single 45 4.79 (1.40) 4.62 (1.12) 0.854 dose of known treatment without oscillations, as in voluntary intake P-value 0.198 0.365 models (Arthington et al., 2014). The components injected are available 3 μ Monocytes (x10 L) 1 0.25 (0.20) 0.21 (0.24) 0.758 directly into the bloodstream without encountering antagonism or 15 0.14 (0.21) 0.18 (0.19) 0.651 30 0.08 (0.13) 0.35 (0.12) 0.045* other interactions (Abuelo et al., 2014; Arthington et al., 2014). The 45 0.17 (0.11) 0.41 (0.14) 0.037* application of injectable minerals showed beneficial health results for P-value 0.625 0.064 calves in the weaning process (Sheffy and Schultz, 1979; Arthington 3 Eosinophils (x10 μL) 1 0.08 (0.11) 0.08 (0.21) 0.901 et al., 2014), as we observed in the present study. Importantly, the 15 0.14 (0.14) 0.01 (0.05) 0.352 calves used in this study were healthy, free of endoparasites, and were 30 0.16 (0.08) 0.07 (0.08) 0.436 fi 45 0.25 (0.23) 0.15 (0.25) 0.621 not trace mineral de cient blood mineral levels within reference values P-value 0.456 0.652 (Villard et al., 2002; Pavlata et al., 2005; Jokubauskienė et al., 2010); therefore, the results discussed below regarding the immune and anti- ≤ Note: results presented in mean and standard deviation. P 0.05 on the same oxidant system were due to the nutraceutical effects of minerals and ff ≤ line shows the di erences between groups. P 0.05 in the same column shows vitamins. the differences over time in each group, the differences being represented by Minerals are fundamental in processes such as growth (Spears, different letters. 2000), and vitamin deficiencies are associated with lower growth rates (Weiss, 2005). In the present study, weight gain did not differ between groups at days 1–45 of the experiment, possibly as a consequence of the

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Fig. 1. Lipoperoxidation (a: LPO), reactive oxygen species (b: ROS), glutathione peroxidase (c: GPx), superoxide dismutase (d: SOD), and total antioxidant capacity against peroxyl radicals (e: ACAP) in the serum of dairy calves receiving subcutaneous vitamins and mineral on days 1 (15 days pre-weaning) and 30 (15 days post- weaning) of experiment. Note: results presented as mean and standard deviation. *P ≤ 0.05 or **P ≤ 0.001 shows the difference between groups. few health challenges faced by calves, as previously described in other those involved in the synthesis of DNA and RNA, responsible for re- studies (Roberts et al., 2016). However, in the final third of the ex- plication and proliferation of immune cells (Andriguetto et al., 1999; periment, there was greater body weight gain in the animals in the Spears, 2000; Roberts et al., 2016). The lower lipid peroxidation levels TREAT group (days 15–30 and days 30–45), when the most critical may also have contributed to increased leukocyte levels (Vedovatto, period in the food transition had passed. Teixeira et al. (2014) observed 2018), because immune cells contain membranes with polyunsaturated no effect on growth performance in nursing dairy calves, and these fatty acids, and these polyunsaturated fatty acids are sensitive to lipid authors cited a number of factors that may play a role in calf perfor- peroxidation by ROS and other free radicals; reduction in lipid damage mance attributable to the major health challenges faced by animals at may indicate minor damage and relative protection of the cells (Spears this stage of life. In a study carried out by our research group, there was and Weiss, 2008). Vitamin E protects cell membranes from lipoperox- a tendency toward greater weight gain in calves during the weaning idation (Andrieu, 2008), acting in synergy with selenium (Mehdi and period (60 days of age) in animals receiving injectable sodium selenite Dufrasne, 2016). and vitamin A and E during the nursing phase (Volpato et al., 2018). Vitamin A acts by neutralizing ROS molecules (Sies, 1991). The Furthermore, when the authors measured body weight of 210-day-old enzymatic antioxidant system is stimulated by the minerals Zn, Cu and calves, they found greater weights in the supplemented group (Weiss, Mn, components of SOD enzymes (Marklund, 1980) and by selenium, 2005). an essential component of the GPx enzyme (Volpato et al., 2018). This The increase in total leukocytes can be explained by the action of explains the activation of SOD and GPx activity over time, reflecting an copper, which stimulates neutrophil and monocyte responses (Maggini increase in total antioxidant capacity (ACAP), as well as lower levels of et al., 2008) and of zinc, a cofactor in more than 300 enzymes including ROS. Other studies have shown that minerals cause decreases in ROS

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Fig. 2. Total protein (a), albumin (b), globulin (c), IgG heavy chain (d), IgA (e) and ceruloplasmin (f) in serum of dairy calves that received mineral and vitamin applications (treated group) by subcutaneous route on days 1 (15 days pre-weaning) and 30 days (15 days post-weaning) of experiment. Note: results presented as mean and standard deviation. *P ≤ 0.05 or **P ≤ 0.001 shows the difference between groups. levels (Santos et al., 2019), as well as increases in antioxidant enzyme overcome basic deficiencies. Increased ceruloplasmin and IgG heavy activity in calves (Glombowsky et al., 2018; Tomasi et al., 2018), cows chains were also observed by Volpato et al. (2018), using a protocol of (Machado et al., 2014) and lambs (Cazarotto et al., 2018), corrobor- association of sodium selenite and vitamins A and E. In the present ating to our results. study, we observed a significant increase in serum levels of TNF-α and We observed that the greater serum concentration of total proteins IL-1 on days 30 and 45 of experiment, as well as in IFNγ on day 45 in was due to increased globulin levels, the basis of the humoral immune animals supplemented with minerals, in disagreement with results re- response that guarantees specificity in the action of antibodies ported by Jiao et al. (2018). According to those authors, copper and (Fernández-Cruz et al., 2009). Similar results were observed by Tomasi zinc reduced intestinal levels of pro-inflammatory cytokines (IL-1 and et al. (2018) and Volpato et al. (2018) who used minerals or a combi- TNF-α) that could be considered an improvement in the immune system nation of minerals and vitamins in nursing calves. The increase in because of a reduction in pro-inflammatory mediators during the globulin levels was caused by elevated levels of IgA and IgG heavy weaning period. Nevertheless, it is important to highlight that slow chain, both of which are important to the immune system (Murata augmentation of IL-1 and TNF-α levels may be positive responses be- et al., 2004), as well as ceruloplasmin, the main copper-chelating pro- cause these cytokines are involved in control of infectious diseases tein in the circulation (Roeser et al., 1970; Sheffy and Schultz, 1979) (Fishman, 1996). As observed in this study, serum levels of IL-1, TNF-α that prevents copper from participating in ROS-producing reactions and IFNγ showed small increases, possibly indicating control of in- (Weiss, 2002; Schneider and Oliveira, 2004). Zinc acts on protein fectious diseases. production and formation of disulfide bonds, present in the structure of In summary, the dairy calves used in this study had no mineral antibodies (Charlton and Ewing, 2007). Deficiencies in selenium and deficiencies (zinc, copper, selenium and manganese) and had no weight copper levels are associated with reduced antibody production and gain differences. All animals remained apparently healthy during the responses to infections (Sheffy and Schultz, 1979; Spears, 2000); experiment, except for one episode of pasty stools in fewer than 5% of therefore, this additional injectable dose in calves is important because the calves (both groups) after withdrawal of the milk from the diet. it may have two important roles as an additional or primary supply to Based on our data, we conclude that mineral complexes, as well as

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Acknowledgments

We thank CAPES, Brazil and CNPq, Brazil for their ﬁnancial support. Thanks also to the State of Santa Catarina for the UNIEDU grant made available to the lead author. Thanks ﬁnally to the Monte Alegre farm for making the animals and facilities available for the study.

References

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